HIV-1 Integrase (IN) is the essential viral protein that inserts the viral genome into host DNA, and is a validated antiviral target. Most recently anti-AIDS drugs that target the active site of IN have been developed and are now in clinical use. However, as is often observed with other drug targets, HIV strains that are resistant to these IN inhibitors have emerged, highlighting the constant need to increase our knowledge of this viral protein so that new inhibitors may be designed to add to our arsenal against this deadly virus. This application proposes to study the molecular structure of HIV IN alone and in combination with viral DNA substrates. To perform integration, IN proteins must multimerize in a particular arrangement with viral DNA in the intasome complex. We reason that understanding the molecular details of how the intasome is formed will reveal new vulnerabilities that can be exploited for developing inhibitors. We propose to improve the physical properties of IN for biophysical studies by introducing sequential targeted changes that will promote intasome formation and stability, while maintaining the catalytic activity required for integration. These changes will be combined to optimize intasome formation and, once isolated, intasomes will be subjected to structure determination by both high and low resolution methods. The structural information gained from the experiments described in this application will lay the foundation for the development of a new class of anti-AIDS drugs that target integrase.